The present invention relates to compositions and methods for isolating one strand of double-stranded nucleic acids.
Many techniques in the field of molecular biology involve hybridizing a target nucleic acid to a support-bound or solution-phase single-stranded oligonucleotide probe for analyzing, capturing, isolating and/or detecting the target nucleic acid. Such techniques range from Sanger-type DNA sequencing (see, e.g., Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467; Ansorge et al., 1987, Nucl. Acids Res. 15:4593-4602; Smith et al., 1985, Nucl. Acids Res. 13:2399-2412; Smith, et al., 1986, Nature 321:674-679; Prober et al., 1987, Science 238:336-341), where a labeled or unlabeled primer is annealed to one strand of a target and enzymatically extended in the presence of 2xe2x80x2,3xe2x80x2-dideoxyribonucleotide terminators (see also, Carrilho, 2000, Electrophoresis 21:55-65 and Kheterpal and Mathies, 1999, Anal. Chem. 71:31Axe2x80x9437A) to array-based gene expression, genotyping, gene mapping and nucleic acid sequencing assays (see, e.g., U.S. Pat. Nos. 5,202,231, 5,695,940 and 5,525,464, WO 95/09248, Khrapko et al., 1991, DNA Sequence 1:375-388; Southern et al., 1992, Genomics 13:1008-1017; Pease et al., 1994, Proc. Natl. Acad. Sci. USA 91:5022-5026; for reviews of the various array-based assays commonly employed in the art see Thompson and Furtado, 1999, Analyst 124:1133-1136; Rockett and Dix, 2000, Xenobiotica 30:155-177; Granjeaud et al., 1999, Bioessays 21:781-790; Lipscutz et al., 1999, Nat. Genet. 21(1 Suppl.):20-24; DeRisi and Iyer, 1999, Curr. Opin. Oncol. 11:76-79; Blanchard, 1998, Genet. Eng. 20:111-123; Case-Green et al., 1998, Curr. Opin. Chem. Biol. 2:404-410; Johnston, 1998, Curr. Biol. 8:R171-174; de Saizieu et al., 1998, Nat. Biotechnol. 16:45-48; and Marshall and Hodgson, 1998, Nat. Biotechnol. 16:27-31).
When the target nucleic acid is single-stranded, hybridization to the oligonucleotide probe occurs relatively easily. But when the target nucleic acid is double-stranded, reassociation of the two target strands strongly competes with, and usually out-competes, hybridization between the target and oligonucleotide probe, especially under the high-salt stringent conditions commonly employed in these assays.
Unfortunately, target nucleic acids are rarely available in single-stranded form. Indeed, most target nucleic acids are double-stranded. For example, genomic DNA, genomic fragments and cDNA are double-stranded. Moreover, one of the most commonly-used amplification techniques for generating analyzable quantities of a specific nucleic acid of interest, the polymerase chain reaction (xe2x80x9cPCRxe2x80x9d see, e.g., U.S. Pat. No. 4,683,202; Sambrook et al., 2d ed. 1989, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Springs Harbor, N.Y.) generates double-stranded amplicons, or target nucleic acids.
Current methods for generating single-stranded target nucleic acids include exonuclease digestion of one strand of the double-stranded target (see, e.g., Hannon et al., 1993, Anal. Biochem. 212:421-427), asymmetric PCR amplification (see, e.g., Stxc3xcrzl and Roth, 1990, Anal. Biochem. 185:164-169), generating single-stranded RNA targets by in vitro transcription of double-stranded PCR amplicons having a T7 or T3 RNA polymerase promoter (see, e.g., Yang and Melera, 1992, BioTechniques 13:922-927) and cloning with Ml 3 (see, e.g., Sambrook, et al., supra). Each of these methods has significant drawbacks that either limit its general applicability and/or significantly increase the time and expense of the assay.
Accordingly, there remains a need in the art for simple methods of generating and isolating one strand of a double-stranded target nucleic acid for assays involving hybridization with a single-stranded oligonucleotide probe, such as Sanger-type sequencing reactions, and array-based mapping, genotyping, expression and sequencing applications.
In one aspect, the present invention provides a method for capturing or isolating one strand of a double-stranded target nucleic acid. The method is based, in part, on the observations that the thermodynamic stabilities and/or kinetics of formation of nucleic acid duplexes under varying conditions of ionic strength depend upon the nature of the internucleoside linkages comprising the strands of the duplex. For example, as evidenced by the occurrence of double-stranded DNAs in nature and the ability of DNAs and RNAs to form homo and hybrid duplexes under physiological conditions, duplexes formed between single-stranded nucleic acids having like-charged internucleoside linkages, such as DNAs and RNAs which have negatively charged sugar phosphodiester interlinkages, are relatively stable under conditions of physiological ionic strength (approximately 100 mM NaCl), temperature (approximately 37xc2x0 C.) and pH (approximately pH 7.2). However, under conditions of low ionic strength (approximately 10 mM NaCl or lower), such DNA/DNA, RNA/RNA and DNA/RNA duplexes tend to dissociate (see, e.g., Egholm et al., 1993, Nature 365:566-568). While not intending to be bound by any particular theory, it is believed that the observed dissociation at conditions of low ionic strength is due to interstrand electrostatic repulsion caused by the negatively charged sugar phosphodiester nucleobase interlinkages.
On the contrary, heteroduplexes in which one strand of the duplex is a conventional DNA or RNA and the other strand is a nucleic acid analog that has a net uncharged or net positively charged backbone (e.g., a PNA) are quite stable at conditions of low ionic strength (see, e.g., id.). In fact, at NaCl concentrations between about 0 and 10 mM, and even as high as 500 mM, such heteroduplexes are significantly more stable than their corresponding DNA/DNA, RNA/RNA or DNA/RNA duplexes (id).
Moreover, the formation of these heteroduplexes is favored kinetically over the formation of the corresponding DNA/DNA, DNA/RNA and RNA/RNA duplexes under the same conditions of low ionic strength. These observed differences in kinetics are independent of the length of the heteroduplex. For example, while a relatively long DNA/DNA target duplex (e.g., xe2x89xa7100 bp) is typically more thermodynamically stable than a short PNA/DNA heteroduplex (e.g., xe2x89xa620 bp) at virtually any ionic strength, if the DNA/DNA target duplex is dissociated and contacted with even a short complementary PNA under conditions of low ionic strength (e.g., less than 10 mM NaCl), the formation of the PNA/DNA heteroduplex will be kinetically favored over the reannealing or reassociation of the DNA target strands. Only as the system is brought to equilibrium will the PNA be displaced by the complementary DNA target strand.
The methods of the invention capitalize on these observed kinetic and thermodynamic stability differences to easily and efficiently isolate one strand of a double-stranded target nucleic acid. Generally, the method involves contacting a double-stranded target nucleic acid with a single-stranded competitor oligonucleotide (xe2x80x9ccompetitor oligoxe2x80x9d) that is capable of hybridizing to one strand of the double-stranded target. The competitor oligo is a nucleic acid analog which comprises a combination of negatively charged (e.g., a native sugar phosphodiester), positively charged (e.g., a sugar glycosyl or positive amide) and/or uncharged (e.g., neutral amide or morpholino-phosphoramidate) nucleobase interlinkages such that the competitor oligo has a net positive charge or a net neutral charge at the desired pH and temperature of use (typically pH 6-9 and 20-40xc2x0 C.). Preferably, the competitor oligo is wholly composed of uncharged nucleobase interlinkages, and optionally includes 3-4 positive interlinkages. The nucleobase sequence of the competitor oligo is at least partially complementary to a portion of one strand of the double-stranded target such that it can hybridize to its complementary target strand.
The target nucleic acid is contacted with the competitor oligo under conditions in which the target strands tend to dissociate from one another and the competitor oligo hybridizes with its complementary target strand, forming a target-strand:competitor oligo heteroduplex and a dissociated target strand. The desired strand of the double-stranded target may then be conveniently recovered by isolating either the dissociated target strand or the heteroduplex using standard isolation and/or capture techniques.
Whether the system is permitted to reach equilibrium prior to isolating the heteroduplex and/or dissociated strand will typically depend upon the thermodynamic stabilities of the double-stranded target and the target-strand:competitor oligo heteroduplex under the condition of the assay, which in turn will depend upon the relative lengths (in nucleobases) of the respective target duplex and heteroduplex. Generally, if the thermodynamic stability (as indicated by thermal melting temperature or Tm) of the target duplex is significantly greater than the thermodynamic stability of the heteroduplex, the system should not be permitted to reach equilibrium. If the thermodynamic stabilities are reversed, or if they are approximately equal, the system may be permitted to reach equilibrium. For example, if the competitor oligo is of a length (in nucleobase units) that is approximately equal to the length of the target nucleic acid, the system may be permitted to reach equilibrium prior to isolating the desired target strand, as the heteroduplex will typically be more thermodynamically stable than the reassociated or reannealed target. However, in instances where the reassociated or reannealed target is thermodynamically more stable than the heteroduplex (such as when the competitor oligo is significantly shorter than the target), the desired strand of the target nucleic acid should be isolated before the system reaches equilibrium.
The method of isolating one strand of a double-stranded target nucleic acid may be carried out in a variety of alternative modes. In one embodiment, the double-stranded target is contacted with the competitor oligo under conditions in which the double-stranded target is stable (i.e., the strands of the target do not readily dissociate). Following contact, the conditions of the mixture are then altered so as to promote dissociation of the target strands and hybridization between the competitor oligo and its complementary target strand.
The conditions may be altered in a single step, or alternatively they may be altered in two or more steps. If a single step is used, the new conditions should simultaneously favor target strand dissociation and target-strand:competitor oligo heteroduplex formation. If a two-step process is used, the conditions are first altered so as to promote target strand dissociation and thereafter altered again so as to promote target-strand:competitor oligo heteroduplex formation.
The conditions may also be cycled. For example, following contact, the temperature of the sample may be cycled between two or more different temperatures. In one convenient embodiment, following contact, the temperature of the sample may be increased to a temperature that is above the thermal melting temperature (Tm) of the double-stranded target and below the Tm of the heteroduplex. The temperature of the sample may then be cycled between this temperature and a second temperature that is above the Tm of the heteroduplex. By choosing a cycling time that takes advantage of the faster kinetics of heteroduplex formation, temperature cycling may be used to drive the dissociation of the double-stranded target and efficient formation of the heteroduplex.
In another embodiment, the strands of the double-stranded target nucleic acid are dissociated prior to contact with the competitor oligo. The conditions used to dissociate the double-stranded target (xe2x80x9cdenaturing conditionsxe2x80x9d) may simultaneously favor target dissociation and target-strand:competitor oligo heteroduplex formation, thereby eliminating the need for further alteration of the conditions. Alternatively, denaturing conditions that do not favor or promote target-strand:competitor oligo heteroduplex formation may be used, and the conditions altered after contact to promote heteroduplex formation. Temperature cycling may be used to drive efficient heteroduplex formation, as previously described.
Regardless of the method used to dissociate the double-stranded target and form the and target-strand:competitor oligo heteroduplex, the dissociated target strand and heteroduplex may be separated from one another and the desired target strand recovered for subsequent use.
The dissociated target strand and target-strand:competitor oligo hetereoduplex may be separated from one another using virtually any technique. For example, the dissociated strand and heteroduplex may be separated by gel electrophoresis, one or both of the respective bands excised from the gel and the nucleic acids eluted from the excised bands. Alternatively, the dissociated target strand and the heteroduplex may be separated from one another by capture. When separated by capture, either the desired target strand or undesired target strand may be captured. If the desired target strand is captured, it may be dissociated and recovered for subsequent use. Capturing the undesired target strand leaves behind the desired target strand, which can be recovered for subsequent use either with or without further purification.
In one embodiment, the dissociated target strand may be hybridized to a complementary oligonucleotide capture probe that includes a capture moiety, such as a biotin, a solid support or a capture sequence, and the hybridized complex isolated by means of the capture moiety. Preferably, the complementary capture probe also comprises nucleobase interlinkages having a net positive or net neutral charge so that the dissociated target strand can be captured under conditions that do not promote reassociation or reannealing of the target strands. Following capture, the complex can be dissociated and the target strand isolated therefrom.
Alternatively, the target-strand:competitor oligo heteroduplex may be isolated by capture. In one convenient embodiment, the competitor oligo includes a capture moiety, such as a biotin, a capture sequence or a solid support, and the heteroduplex is isolated from the dissociated target strand by means of the capture moiety. For example, if the competitor oligo includes a biotin capture moiety, the heteroduplex may be isolated from the dissociated target strand by contacting the sample with immobilized streptavidin, for example by passing the sample through a column packed with streptavidin-coated beads or by contacting the sample with straptavidin-coated magnetic beads, which can be removed with the aid of a magnet. If the competitor oligo includes a capture sequence, the heteroduplex may be isolated from the dissociated target strand via a complementary capture probe that also includes a capture moiety, such as a biotin or a solid support. The capture probe may be an RNA or a DNA oligomer or, like the competitor oligo, it may comprise a backbone having a net positive or net neutral charge at the pH and temperature of the assay. When the competitor oligo includes a solid support capture moiety, the heteroduplex may be isolated from the dissociated target strand by, e.g., filtration, decanting, etc. If the solid support is magnetic, the heteroduplex may be conveniently isolated with the aid of a magnet.
In another embodiment, the heteroduplex is isolated by capture with a capture probe capable of forming a triplex with the heteroduplex. Preferably, the triplex capture probe is modified to include a capture moiety, such as biotin or a solid support, as previously described. In one convenient embodiment, the triplex-forming capture probe is a PNA (see, e.g., U.S. Pat. Nos. 5,986,053; 5,641,625; and 5,539,082, which are incorporated herein by reference).
In another aspect, the invention provides kits for practicing the methods of the invention. Generally, the kits comprise a competitor oligo complementary to at least a portion of one strand of a target nucleic acid of interest and buffers useful for dissociating the double-stranded target and effecting formation of a target-strand:competitor oligo heteroduplex. The kit may also comprise capture probes or other means for isolating either the dissociated target strand or heteroduplex, or both. The competitor oligo may be optionally modified with a capture moiety to facilitate capture of the target-strand:competitor oligo heteroduplex, as previously described. Alternatively, the kit may further comprise reagents useful for modifying the competitor oligo, a capture probe or both, with a capture moiety.
Target strands isolated according to the methods and kits of the invention may be further manipulated using techniques well known in the art. For example, the isolated strand may be used as a template in a subsequent PCR amplification, as a target for solution-phase and/or array-based sequencing by hybridization, mapping, gene expression and genotyping assays, or as a template in a Sanger-type sequencing reaction.